The differential polarized distribution of the reducedfolate transporter (RFT-1) and folate receptor ␣ (FR␣), the two proteins involved in the transport of folate, has been characterized in normal mouse retinal pigment epithelium (RPE) and in cultured human RPE cells. RPE cells mediate the vectorial transfer of nutrients from choroidal blood to neural retina. Whereas FR␣ is known to be present in many cell types of the neural retina, in situ hybridization analysis in the present study demonstrated that RFT-1 is present only in RPE. Laser-scanning confocal microscopy using antibodies specific for RFT-1 demonstrated an apical distribution of this protein in cultured human and intact mouse RPE, which contrasts with the basolateral distribution of FR␣ in these cells. The expression of RFT-1 in the RPE cell apical membrane was confirmed by functional studies with purified apical membrane vesicles from bovine RPE. These studies, done with N 5 -methyltetrahydrofolate (the predominant folate derivative in blood) and folate as substrates, have shown that RFT-1 functions in a Na ؉ -and C1؊ -independent manner. The transporter is specific for folate and its analogs. A transmembrane H ؉ gradient influences the transport function of this protein markedly; the transport mechanism is likely to be either folate/H ؉ co-transport or folate/OH ؊ exchange. Based on the differential polarization of FR␣ and RFT-1 in RPE, we suggest that these two proteins work in a concerted manner to bring about the vectorial transfer of folate across the RPE cell layer from the choroidal blood to the neural retina. This constitutes the first report of the differential polarization of the two folate transport proteins in any polarized epithelium.The one-carbon derivatives of the water-soluble vitamin folic acid are essential for intermediary metabolism. These derivatives are required for the synthesis of purine and pyrimidine nucleotide precursors of RNA and DNA and also for metabolism of several amino acids. Since folate and its one-carbon

In situ hybridization analysis showed that OCT3 is expressed in mouse RPE and in several cell types of the neural retina, including photoreceptor, ganglion, amacrine, and horizontal cells. The expression of OCT3 in RPE was confirmed by Northern blot analysis and RT-PCR. The characteristics of MPP( +) uptake in cultured ARPE-19 cells included the stimulation of transport by alkaline pH, high affinity (K(t) = 28 +/- 4 microM), competition with several cationic drugs and monoamine neurotransmitters and sensitivity to steroids. In addition, the uptake of MPP(+) in RPE cells was inhibited by dopamine and histamine with IC(50) values (concentration needed for 50% inhibition) of 637 +/- 84 microM and 150 +/- 20 microM, respectively. CONCLUSIONS. This study provides the first report on the expression and function of an organic cation transporter, OCT3, in the eye and in particular the RPE. The data have physiological and pharmacological relevance as it is likely that OCT3 participates in the clearance of dopamine and histamine from the subretinal space and may also play a key role in the disposition of the retinal neurotoxin MPP(+).

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We describe here the cloning and functional characterization of an organic cation transporter from Caenorhabditis elegans (CeOCT1). The CeOCT1 cDNA is 1826 bp long and codes for a protein of 568 amino acids. The oct1 gene is approximately 3.2 kb in size and consists of 12 exons. The location of this gene corresponds to the F52F12.1 gene locus on chromosome I. The predicted protein contains 12 putative transmembrane domains. It exhibits significant homology to mammalian OCTs. When expressed in mammalian cells, CeOCT1 induces the transport of the prototypical organic cation tetraethylammonium. The Michaelis-Menten constant for this substrate is 80+/-16 microM. The substrate specificity of CeOCT1 is broad. This represents the first report on the cloning and functional characteristics of an organic cation transporter from C. elegans.

In situ hybridization analysis showed that OCT3 is expressed in mouse RPE and in several cell types of the neural retina, including photoreceptor, ganglion, amacrine, and horizontal cells. The expression of OCT3 in RPE was confirmed by Northern blot analysis and RT-PCR. The characteristics of MPP( +) uptake in cultured ARPE-19 cells included the stimulation of transport by alkaline pH, high affinity (K(t) = 28 +/- 4 microM), competition with several cationic drugs and monoamine neurotransmitters and sensitivity to steroids. In addition, the uptake of MPP(+) in RPE cells was inhibited by dopamine and histamine with IC(50) values (concentration needed for 50% inhibition) of 637 +/- 84 microM and 150 +/- 20 microM, respectively. CONCLUSIONS. This study provides the first report on the expression and function of an organic cation transporter, OCT3, in the eye and in particular the RPE. The data have physiological and pharmacological relevance as it is likely that OCT3 participates in the clearance of dopamine and histamine from the subretinal space and may also play a key role in the disposition of the retinal neurotoxin MPP(+).